The cultures were prepared by inoculating 80 mL of PDY broth in 250-mL Erlenmeyer flask with 8 mL of 5-day-old pre-inocula. When indicated, CuSO4 (150 μM) was added to the cultures. The GenBank accession number of the sequence of the P. ostreatus laccase poxa1b gene (Giardina et al., 1999) reported in this paper
is AJ005017. To prepare the vector pEGFPea1b (Fig. 1a) designed to study the poxa1b promoter through enhanced GFP gene expression in P. ostreatus, the poxa1b terminator and the poxa1b promoter were amplified by PCR using plasmid vectors selected from the P. ostreatus genomic library (Giardina et al., 1995, 1996, 1999) as templates and the gene-specific oligonucleotides Termpoxa1bXbaI/Termpoxa1bPstI and Prompoxa1SacIrev/Prompoxa1SacIfw (Table 1) as primers, respectively. The amplified fragment of poxa1b terminator was subjected to hydrolysis with the restriction selleck chemicals llc enzymes XbaI and PstI and ligated TSA HDAC into the XbaI-/PstI-digested pUC13 vector, giving the vector pA1BTERM. An intron/exon fragment was prepared by annealing of the synthetic oligomers EGFP1dir and EGFP1rev having complementary sequences including poxc gene intron number XIX flanked by two
amino acids at the 5′ end and three at 3′ end (Giardina et al., 1996) and the sticky ends features of the restriction enzymes SacI and BamHI, followed by digestion by SacI and BamHI. The egfp gene was amplified by PCR using the plasmid vector pEGFP-C1 (Clontech Laboratories, Inc., CA) as template and the gene-specific oligonucleotides EGFP3dir/EGFP5rev Methocarbamol as primers (Table 1). The plasmid vector pA1BTERM was subjected
to hydrolysis by the endonucleases SacI and XbaI, and ligation reaction among the amplified egfp gene, the intron/exon fragment, and the linearized pA1BTERM vector was carried out. The vector thus obtained was subjected to SacI/EcoRI hydrolysis and ligated to the amplified poxa1b promoter fragment after SacI/EcoRI digestion, giving the pEGFPea1b vector. The vector pEGFPCBX (Fig. 1b) was constructed by cloning the DNA fragment resulting from NotI/SphI hydrolysis of pTM1 into pEGFPea1b vector. This fragment includes the gene cbxR and its own promoter and terminator. To include the desired restriction sites NotI/SphI within pEGFPea1b, this vector was hydrolyzed by the enzymes SphI–EcoRI, and an oligonucleotide whose sequence contains the polylinker EcoRI–NotI–SphI was then ligated. Ligation between the DNA fragment excised from pTM1 and pEGFPea1b hydrolyzed by NotI and SphI was then carried out. Liquid cultures of P. ostreatus for protoplasting were set up by inoculating 60 mL YMG broth [1% glucose, 0.4% yeast extract (Difco), 1% malt extract] in 250-mL cotton plugged Erlenmeyer flasks with six agar plugs (11 mm diameter) of P. ostreatus mycelium, grown on PDA [2.4% potato dextrose (Difco)] medium. The inocula were incubated in a temperature-controlled incubator at 28 °C on a rotary shaker (at 120 rpm).